Harm Wopereis

Altered gut microbiota and activity in a murine model of autism spectrum disorders.

Autism spectrum disorder (ASD) is a heterogeneous group of complex neurodevelopmental disorders with evidence of genetic predisposition. Intestinal disturbances are reported in ASD patients and compositional changes in gut microbiota are described. However, the role of microbiota in brain disorders is poorly documented. Here, we used a murine model of ASD to investigate the relation between gut microbiota and autism-like behaviour. Using next generation sequencing technology, microbiota composition was investigated in mice in utero exposed to valproic acid (VPA). Moreover, levels of short chain fatty acids (SCFA) and lactic acid in caecal content were determined. Our data demonstrate a transgenerational impact of in utero VPA exposure on gut microbiota in the offspring. Prenatal VPA exposure affected operational taxonomic units (OTUs) assigned to genera within the main phyla of Bacteroidetes and Firmicutes and the order of Desulfovibrionales, corroborating human ASD studies. In addition, OTUs assigned to genera of Alistipes, Enterorhabdus, Mollicutes and Erysipelotrichalis were especially associated with male VPA-exposed offspring. The microbial differences of VPA in utero-exposed males deviated from those observed in females and was (i) positively associated with increased levels of caecal butyrate as well as ileal neutrophil infiltration and (ii) inversely associated with intestinal levels of serotonin and social behaviour scores. These findings show that autism-like behaviour and its intestinal phenotype is associated with altered microbial colonization and activity in a murine model for ASD, with preponderance in male offspring. These results open new avenues in the scientific trajectory of managing neurodevelopmental disorders by gut microbiome modulation.

Improved Detection of Bifidobacteria with Optimised 16S rRNA-Gene Based Pyrosequencing

The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, ‘bifidobacteria-optimised’ universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.

The first thousand days - intestinal microbiology of early life: establishing a symbiosis

The development of the intestinal microbiota in the first years of life is a dynamic process significantly influenced by early‐life nutrition. Pioneer bacteria colonizing the infant intestinal tract and the gradual diversification to a stable climax ecosystem plays a crucial role in establishing host–microbe interactions essential for optimal symbiosis. This colonization process and establishment of symbiosis may profoundly influence health throughout life. Recent developments in microbiologic cultivation‐independent methods allow a detailed view of the key players and factors involved in this process and may further elucidate their roles in a healthy gut and immune maturation. Aberrant patterns may lead to identifying key microbial signatures involved in developing immunologic diseases into adulthood, such as asthma and atopic diseases. The central role of early‐life nutrition in the developmental human microbiota, immunity, and metabolism offers promising strategies for prevention and treatment of such diseases. This review provides an overview of the development of the intestinal microbiota, its bidirectional relationship with the immune system, and its role in impacting health and disease, with emphasis on allergy, in early life.

Influence of fermented milk products, prebiotics and probiotics on microbiota composition and health

The gut microbiota is a highly diverse and relative stabile ecosystem increasingly recognized for its impact on human health. The homeostasis of microbes and the host is also referred to as eubiosis. In contrast, deviation from the normal composition, defined as dysbiosis, is often associated with localized diseases such as inflammatory bowel disease or colonic cancer, but also with systemic diseases like metabolic syndrome and allergic diseases. Modulating a gut microbiota dysbiosis with nutritional concepts may contribute to improving health status, reducing diseases or disease symptoms or supporting already established treatments. The gut microbiota can be modulated by different nutritional concepts, varying from specific food ingredients to complex diets or by the ingestion of particular live microorganisms. To underpin the importance of bacteria in the gut, we describe molecular mechanisms involved in the crosstalk between gut bacteria and the human host, and review the impact of different nutritional concepts such as pre-, pro- and synbiotics on the gastrointestinal ecosystem and their potential health benefits. The aim of this review is to provide examples of potential nutritional concepts that target the gut microbiota to support human physiology and potentially health outcomes.

Size and phospholipid coating of lipid droplets in the diet of young mice modify body fat accumulation in adulthood

Background:In addition to contemporary lifestyle factors that contribute to the increased obesity prevalence worldwide, early nutrition is associated with sustained effects on later life obesity. We hypothesized that physical properties of dietary lipids contribute to this nutritional programming. We developed a concept infant formula (IMF) with large, phospholipid-coated lipid droplets (Nuturis; Danone Research, Paris, France) and investigated its programming effect on metabolic phenotype later in life.Methods:Male C57Bl/6j mice were fed a control formula (Control IMF) or Nuturis (Concept IMF) diet between postnatal day (PN)16 and PN42. All mice were subsequently fed a Western-style diet (WSD) until PN126. Body composition was monitored repeatedly by dual-energy X-ray absorptiometry between PN42 and PN126.Results:Concept IMF slightly increased lean body mass as compared with Control IMF at PN42 but did not affect fat mass. Upon 84 d of WSD feeding, the Concept IMF group showed reduced fat accumulation as compared with Control IMF. In addition, fasting plasma leptin, resistin, glucose, and lipids were significantly lower in the Concept IMF group.Conclusion:Large phospholipid-coated lipid droplets in young mice reduced fat accumulation and improved metabolic profile in adulthood. These data emphasize that physical properties of early dietary lipids contribute to metabolic programming.

Synbiotics‐supplemented amino acid‐based formula supports adequate growth in cow's milk allergic infants

Children with cows milk allergy (CMA) are at risk for inadequate nutritional intake and growth. Dietary management of CMA, therefore, requires diets that are not only hypoallergenic but also support adequate growth in this population. This study assessed growth of CMA infants when using a new amino acid‐based formula (AAF) with prebiotics and probiotics (synbiotics) and evaluated its safety in the intended population.

Intestinal microbiota in infants at high risk for allergy: Effects of prebiotics and role in eczema development

Background: Development of the gut microbiota in infancy is important in maturation of the immune system. Deviations in colonization patterns have been associated with allergic manifestations such as eczema, but exact microbiome dysfunctions underlying allergies remain unclear. We studied the gut microbiota of 138 infants at increased risk of allergy, participating in a clinical trial investigating the effectiveness of a partially hydrolyzed protein formula supplemented with nondigestible oligosaccharides on the prevention of eczema. Objective: The effects of interventions and breast‐feeding on fecal microbiota were investigated. Additionally, we aimed to identify microbial patterns associated with the onset of eczema. Methods: Bacterial taxonomic compositions in the first 26 weeks of life were analyzed by using 16S rRNA gene sequencing. Additionally, fecal pH and microbial metabolite levels were measured. Results: Fecal microbial composition, metabolites, and pH of infants receiving partially hydrolyzed protein formula supplemented with nondigestible oligosaccharides was closer to that of breast‐fed infants than that of infants receiving standard cows milk formula. Infants with eczema by 18 months showed discordant development of bacterial genera of Enterobacteriaceae and Parabacteroides species in the first 26 weeks, as well as decreased acquisition of lactate‐utilizing bacteria producing butyrate, namely Eubacterium and Anaerostipes species, supported by increased lactate and decreased butyrate levels. Conclusions: We showed that a partially hydrolyzed protein infant formula with specific prebiotics modulated the gut microbiota closer to that of breast‐fed infants. Additionally, we identified a potential link between microbial activity and onset of eczema, which might reflect a suboptimal implementation of gut microbiota at specific developmental stages in infants at high risk for allergy. GRAPHICAL ABSTRACT Figure. No caption available.

A synbiotic-containing amino acid-based formula improves gut microbiota in non-IgE-mediated allergic infants

BackgroundPrebiotics and probiotics (synbiotics) can modify gut microbiota and have potential in allergy management when combined with amino-acid-based formula (AAF) for infants with cow’s milk allergy (CMA).MethodsThis multicenter, double-blind, randomized controlled trial investigated the effects of an AAF-including synbiotic blend on percentages of bifidobacteria and Eubacterium rectale/Clostridium coccoides group (ER/CC) in feces from infants with suspected non-IgE-mediated CMA. Feces from age-matched healthy breastfed infants were used as reference (healthy breastfed reference (HBR)) for primary outcomes. The CMA subjects were randomized and received test or control formula for 8 weeks. Test formula was a hypoallergenic, nutritionally complete AAF including a prebiotic blend of fructo-oligosaccharides and the probiotic strain Bifidobacterium breve M-16V. Control formula was AAF without synbiotics.ResultsA total of 35 (test) and 36 (control) subjects were randomized; HBR included 51 infants. At week 8, the median percentage of bifidobacteria was higher in the test group than in the control group (35.4% vs. 9.7%, respectively; P<0.001), whereas ER/CC was lower (9.5% vs. 24.2%, respectively; P<0.001). HBR levels of bifidobacteria and ER/CC were 55% and 6.5%, respectively.ConclusionAAF including specific synbiotics, which results in levels of bifidobacteria and ER/CC approximating levels in the HBR group, improves the fecal microbiota of infants with suspected non-IgE-mediated CMA.

Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis

BackgroundPerturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn−/− mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn−/− mice.ResultsUsing high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn−/− mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn−/− mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn−/− mice. The arthritis phenotype in IL1rn−/− mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn−/− microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis.ConclusionsThese findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.

Cross-feeding between Bifidobacterium infantis and Anaerostipes caccae on lactose and human milk oligosaccharides

The establishment of the gut microbiota immediately after birth is a dynamic process that may impact lifelong health. At this important developmental stage in early life, human milk oligosaccharides (HMOS) serve as specific substrates to promote the growth of gut microbes, particularly the group of Actinobacteria (bifidobacteria). Later in life, this shifts to the colonisation of Firmicutes and Bacteroidetes, which generally dominate the human gut throughout adulthood. The well-orchestrated transition is important for health, as an aberrant microbial composition and/or SCFA production are associated with colicky symptoms and atopic diseases in infants. Here, we study the trophic interactions between an HMOS-degrader, Bifidobacterium longum subsp. infantis and the butyrogenic Anaerostipes caccae using carbohydrate substrates that are relevant in this early life period, i.e. lactose and HMOS. Mono-and co-cultures of these bacterial species were grown at pH 6.5 in anaerobic bioreactors supplemented with lactose or total human milk carbohydrates (containing both lactose and HMOS). A cac was not able to grow on these substrates except when grown in co-culture with B. inf, leading concomitant butyrate production. Cross-feeding was observed, in which A. cac utilised the liberated monosaccharides as well as lactate and acetate produced by B. inf. This microbial cross-feeding is indicative of the key ecological role of bifidobacteria in providing substrates for other important species to colonise the infant gut. The symbiotic relationship between these key species contributes to the gradual production of butyrate early in life that could be important for host-microbial cross-talk and gut maturation. Importance The establishment of a healthy infant gut microbiota is crucial for the immune, metabolic and neurological development of infants. Recent evidence suggests that an aberrant gut microbiota early in life could lead to discomfort and predispose infants to the development of immune related diseases. This paper addresses the ecosystem function of two resident microbes of the infant gut. The significance of this research is the proof of cross-feeding interactions between HMOS-degrading bifidobacteria and a butyrate-producing microorganism. Bifidobacteria in the infant gut that support the growth and butyrogenesis of butyrate-producing bacteria, could orchestrated an important event of maturation for both the gut ecosystem and physiology of infant.